SphK1-driven autophagy potentiates focal adhesion paxillin-mediated metastasis in colorectal cancer

Abstract

Invasion and metastasis are the main causes of colorectal cancer (CRC)-related death. Accumulating evidence suggested that sphingosine kinase 1 (SphK1) promoted the metastasis of CRC and autophagy played an important role in SphK1 promoting the metastasis of malignancy. However, the mechanism by which SphK1-driven autophagy promotes invasion and metastasis in CRC remains to be clarified. In the present study, immunohistochemical detection showed the expression of SphK1 and paxillin was higher in human CRC tissues than those of normal colorectal mucosal tissues, they were both associated with TNM staging, lymphatic, and distance metastasis. In addition, study of in situ tumor transplantation model in nude mice showed that the suppression of SphK1 inhibited the growth of colonic orthotopic implantation tumors and the expression of paxillin, p-paxillin, LC3 in the tumor. So, SphK1 may promote CRC metastasis via inducing the expression of paxillin expression and its phosphorylation, in vivo. Furthermore, results of CCK8 assay, transwell and wound healing assays showed that SphK1 promoted the viability, invasion, and metastasis of CRC cells. Transmission electron microscopy detection showed that SphK1 is the key factor in autophagy induction in CRC cells. Moreover, western blot examination indicated that the expression of LC3Ⅱ/Ⅰ, paxillin, p-paxillin, MMP-2, and vimentin was enhanced in SphK1-overexpressed CRC cells and suppressed in SphK1 knockdown CRC cells, meanwhile, the expression of E-cadherin was suppressed in SphK1-overexpressed CRC cells and enhanced in SphK1 knockdown CRC cells. Suppression of autophagy by 3MA reversed the expression of paxillin and its phosphorylation in SphK1-overexpressed CRC cells, indicated that SphK1-driven autophagy induced the expression of paxillin and its phosphorylation in CRC cells. Together, these findings reveal that SphK1-driven autophagy may promote the invasion and metastasis of CRC via promoting the expression of focal adhesion paxillin and its phosphorylation.

Keywords: autophagy; colorectal cancer; metastases; paxillin; sphingosine kinase 1.

FIGURE 1

Aberrant expression of SphK1 and paxillin in CRC. Immunohistochemical staining of SphK1 and paxillin in CRC and normal colorectal mucosal tissues (magnification, ×400, *p < 0.05 vs. normal or non‐metastasis group). Data are shown as the mean ± SD of three replicates.

FIGURE 2

SphK1 was successfully constructed in CRC cells. qRT‐PCR revealing SphK1 mRNA expression in RKO and HT29 cells. The relative expression of SphK1 was quantified via normalization to GAPDH. (**p < 0.01 vs. NC‐RKO or NC‐HT29 group). Data are shown as the mean ± SD of three replicates.

FIGURE 3

Overexpression of SphK1 significantly promoted viability of HT29 cells. CCK8 assay of CRC cell viability in vitro. (**p < 0.01 vs. NC‐RKO or NC‐HT29 group). Data are shown as the mean ± SD of three replicates.

FIGURE 4

Overexpression of SphK1 promote migration and metastasis of CRC cells. Wound healing assay showing migrated RKO and HT29 cells transfected with shSphK1 or overexpression SphK1. (magnification, ×40, *p < 0.05 or **p < 0.01 vs. NC‐RKO or NC‐HT29 group). Data are shown as the mean ± SD of three replicates.

FIGURE 5

Overexpression of SphK1 promote migration and metastasis of CRC cells. Transwell assay of showing migrated RKO and HT29 cells transfected with shSphK1 or overexpression SphK1. (magnification, ×200, **p < 0.01 vs. NC‐RKO or NC‐HT29 group). Data are shown as the mean ± SD of three replicates.

FIGURE 6

SphK1 is involved in induced autophagy in CRC cells. Transmission electron microscopy showing formation of autophagy flux after transfected with shSphK1 or overexpression SphK1 in RKO and HT29 cells. The arrow shows the autophagosomes/autolysosomes. (*p < 0.05, **p < 0.01 vs. NC‐RKO or NC‐HT29 group). Data are shown as the mean ± SD of three replicates.

FIGURE 7

SphK1 facilitates autophagy, EMT factors, and focal adhesions pathway. (A, B) Western blot analysis of SphK1, LC3Ⅱ/Ⅰ, paxillin, p‐paxillin, MMP2, E‐cadherin, and vimentin in CRC cells. (*p < 0.05 vs. NC‐RKO group, *p < 0.05 vs. NC‐HT29. (C) Western blot revealing LC3, paxillin, and p‐paxillin after 3MA treatment (10 mM, 24 h) in SphK1(+)‐HT29. (*p < 0.05 vs. NC‐HT29 group, SphK1(+)‐HT29 group or NC‐HT29 group +3MA). Data are shown as the mean ± SD of three replicates.

FIGURE 8

The silencing of SphK1 in RKO cells leads to delayed growth of tumor in nude mice. (A) The orthotopic transplantation nude mice in SphK1(‐)‐RKO and NC‐RKO groups. (B) The size of orthotopic tumors in transplanted nude mice in SphK1(‐)‐RKO and NC‐RKO groups. (**p < 0.01 vs. NC‐RKO group). Data are presented as mean ± SD (n = 5).

FIGURE 9

Low expression of SphK1, LC3, paxillin, and p‐paxillin in SphK1(‐)‐RKO. Immunohistochemical staining of SphK1, paxillin, p‐paxillin, and LC3 in orthotopic tumor tissues. (magnification, ×400, *p < 0.05 vs. NC‐RKO group). Data are shown as the mean ± SD of three replicates.

FIGURE 10

Schematic model showing the role of SphK1 in the regulation of EMT progression and FA signaling.